A SIMPLE METHOD FOR QUANTIFYING CHANGES IN NEURONAL POPULATIONS IN PRIMARY CULTURES OF DISSOCIATED RAT-BRAIN

A simple method which allows easy quantification of neuronal and glial sub-populations in primary neuronal tissue culture is described. The method takes advantage of the fact that the staining of neuronal and g lia nuclei with Hoechst 33258 is distinctive and characteristic of the cell type. Double-staining experiments show that only cells with nucl ear staining characteristic of neurones are immunopositive for neurona l markers. There is no obvious difference between neurones from differ ent regions of the brain nor any apparent differences in nuclear stain ing of neurones of different sizes. This method is particularly useful for the quantification of neurones in cultures where neurones are gro wing on glial beds or where the neurones are growing in clusters. Furt her, it eliminates the need to double-stain cultures with neuronal mar kers in order to calculate neuronal sub-populations. We have used this technique to examine the effect of time in vitro on the proportion of calbindin D-28K-positive neurones in striatal cultures. We show that with increasing time in culture, there is a significant increase in th e percentage of neurones which express calbindin D-28K. This supports the suggestion that calbindin D-28K may be important for promoting the survival of neurones in which it is expressed.